Diet and the colonic exfoliome: a novel, non-invasive approach to testing interventions in humans

项目总结 饮食是结直肠癌(CRC)的重要危险因素，几种饮食成分与 结直肠癌是通过肠道微生物代谢进行修饰的。微生物发酵膳食纤维产生短链 脂肪酸，例如，醋酸盐、丙酸和丁酸。膳食纤维已被证明可以减少结肠癌 在动物模型和体外实验中，丁酸盐影响对癌症风险至关重要的细胞通路。更进一步，从 我们的小组认为丁酸和omega-3多不饱和脂肪酸(n-3PUFA)的联合作用 可能会增强这些饮食成分的化学预防潜力。我们假设相对较低的 西方人群中n-3多不饱和脂肪酸和纤维的摄入量以及它们之间的相互作用 饮食成分可以解释为什么n-3多不饱和脂肪酸和可发酵纤维的化学保护作用没有 在前瞻性队列研究中一直被检测到。我们假设，长期的- 补充链n-3多不饱和脂肪酸和补充高度可发酵的纤维将改变关键途径 对预防结直肠癌很重要，特别是由线粒体介导的细胞程序性死亡 由于脂质活性氧的积累(铁下垂)，细胞增殖，以及 二十烷类/炎症途径。 我们建议在30名健康男性和女性(50-75岁)中进行随机、对照交叉试验研究，以 比较补充可溶性纤维(35 g/d)+补充n-3多不饱和脂肪酸(6 g/d EPA+DHA)的数量 镜像意味着每天的摄入量与较低的CRC风险相关，麦芽糊精和玉米油控制。大便 样本将在两次为期30天的干预的开始、中期(第15天)和结束时收集 句号。使用一种新的、经济有效的、非侵入性的方法，我们将评估全球基因的差异。 利用RNA-Seq技术在大便脱落层(即大便中脱落的结肠上皮细胞)中的表达特征。 我们将集中在与结肠细胞增殖和凋亡/铁性下垂、细胞表型和 炎症反应。此外，我们将评估与丁酸盐有关的肠道微生物功能基因的变化。 采用液滴数字聚合酶链式反应(DdPCR)进行扩增。在干预期间收集多个样本 将提供应对补充n-3多不饱和脂肪酸的纵向变化的关键衡量标准，并 可发酵纤维。 这项拟议的试验性人类机械学研究将是第一个在人类中整合和表征 N-3多不饱和脂肪酸与可发酵纤维暴露的关系 其他与结直肠癌相关的细胞信号通路通过肠道微生物组的代谢活动。这样做的结果是 受控干预将有助于将当前的机制知识从临床前动物模型转化为 对人类和消除风险，并告知预防儿童权利公约的方法。审问大便脱落是一种 研究干预措施对人体肠道影响的新的、经济有效的、非侵入性的方法。

PROJECT SUMMARY Diet is an important risk factor for colorectal cancer (CRC) and several dietary constituents implicated in CRC are modified by gut microbial metabolism. Microbial fermentation of dietary fiber produces short chain fatty acids, e.g., acetate, propionate, and butyrate. Dietary fiber has been shown to reduce colon tumors in animal models and, in vitro, butyrate influences cellular pathways important to cancer risk. Further, work from our group suggests that the combined effects of butyrate and omega-3 polyunsaturated fatty acids (n-3 PUFA) may enhance the chemopreventive potential of these dietary constituents. We postulate that the relatively low intakes of n-3 PUFA and fiber in Western populations and the failure to address interactions between these dietary components may explain why chemoprotective effects of n-3 PUFA and fermentable fibers have not been detected consistently in prospective cohort studies. We hypothesize that the combined effects of long- chain n-3 PUFA supplementation and supplemental highly fermentable fiber will alter critical pathways important to CRC prevention, particularly intrinsic mitochondrial-mediated programmed cell death resulting from the accumulation of lipid reactive oxygen species (ferroptosis), cell proliferation, and the eicosanoid/inflammation pathways. We propose a randomized, controlled crossover pilot study in 30 healthy men and women (50-75 y) to compare supplemental soluble fiber (35 g/d) + supplemental n-3 PUFA (6 g/d EPA+DHA), in quantities mirroring mean daily intakes associated with lower CRC risk, with a maltodextrin and corn oil control. Stool samples will be collected at the beginning, middle (day 15), and end of each of the two 30-day intervention periods. Using a novel, cost-effective, non-invasive approach, we will evaluate differences in global gene expression signatures in the stool exfoliome (i.e., sloughed colonic epithelial cells in stool) using RNA-Seq. We will focus on pathways related to colonic cell proliferation and apoptosis/ferroptosis, cell phenotype, and inflammatory response. Further, we will evaluate changes in gut microbial functional genes involved in butyrate production using droplet digital PCR (ddPCR). The collection of multiple samples over the intervention periods will provide a critical measure of longitudinal changes in response to the supplemental n-3 PUFA and fermentable fiber. This proposed pilot human mechanistic study will be the first in humans to integrate and characterize the relationships between n-3 PUFA and fermentable fiber exposure that modulate programmed cell death and other CRC-related cell-signaling pathways through metabolic activities of the gut microbiome. Results of this controlled intervention will help to translate the current mechanistic knowledge from preclinical animal models to humans and to de-risk and inform approaches for CRC prevention. Interrogating the stool exfoliome is a novel, cost-effective, non-invasive approach to studying effects of interventions on the human gut.